1. Introduction
Protein ubiquitination is a reversible covalent modification that regulates the activity, localization, and stability of target proteins. While generally considered a terminate signal for proteasomal degradation, ubiquitination is also appreciated for its functions in protein modulation, including receptor down-modulation, protein–protein interactions, and gene transcription [
1]. Protein ubiquitination has also emerged as a critical mechanism of immune response regulation, including lymphocyte development and activation, intracellular signal transduction, antigen presentation, and immune evasion [
2,
3]. Recent work has shown that various ubiquitin (Ub) ligases help to prevent the immune system from attacking self-tissues, implicating the dysfunction of Ub in autoimmune diseases [
2,
4].
Ub-activating (E1) enzymes catalyze the first step in the ubiquitination cascade, which results in the addition of Ub and other Ub-like proteins to target proteins. Besides UBE1 (Ub-activating enzyme 1 [UBA1]), Ub-activating enzyme 6 (UBA6) is an alternative enzyme for Ub activation [
5,
6,
7,
8]. Mice lacking UBA6 died at E5.5 of embryonic development, indicating the importance of UBA6 during embryogenesis [
5,
9]. The physiological functions of UBA6 remain poorly understood; however, we have previously reported that UBA6 is required for the viability of mouse embryo fibroblasts (MEFs), and loss of neuronal UBA6 resulted in abnormal hippocampal and amygdala development [
9,
10,
11,
12]. UBA6 is abundantly expressed in many organs, including the thymus and peripheral lymphoid organs, raising the question of UBA6 function in T cells. The distinguishing feature of UBA6 from UBA1 is that it is required for activation of the E2-conjugating enzyme USE1 (also referred to as UBE2Z), a lysine-48 chain linkage-specific E2 [
10]. Of the approximately two dozen E2 enzymes encoded by the human genome, USE1 relies solely on UBA6 for charging [
7,
10]. Moreover, to date, USE1 has been shown to function together with UBR1, UBR2, and UBR3 [
10,
13]. These E3s constitute primary members of the N-end rule class of E3s, and in this context, the UBA6–USE1–UBR pathway has been shown to control the turnover of RGS proteins [
10]. However, this pathway likely controls the turnover of additional proteins through other E3 ligases.
Ub has a central role in the regulation of several pathways leading to the activation of NF-κB, which is vital for both innate and adaptive immunity [
2]. NF-κB is necessary for T cell activation, survival, and proliferation, as well as effective immune responses [
2,
14]. Given the established roles of Ub and NF-κB in T cell responses, we hypothesized that UBA6 could be involved in the activation and function of immune cells. We induced immune cell activation to test this hypothesis and found that UBA6 levels were elevated in T cells only. Subsequently, we generated T cell-specific conditional UBA6-deficient mice under the control of the proximal lck promoter and evaluated the function of UBA6 in T cell activation. We found that deletion of UBA6 in T cells greatly increased differentiation and proliferation in immune stimulation. This function of UBA6 was mediated by destabilized IκB degradation, resulting in increased activation of NF-κB p65. These data indicated that alteration of the UBA6 pathway in T cells leads to defects in T cell response homeostasis and demonstrates a novel role for UBA6 in controlling T cell receptor (TCR) signal transduction pathways.
2. Materials and Methods
2.1. Mice
UBA6
flox/flox mice have been previously described [
9,
12]. Lck-Cre mice and RAG1
−/− mice were obtained from the Jackson Laboratory and C57BL6 mice were purchased from Hychang Science (Daegu, Korea). Mice were then bred at the animal facilities of the Asan Institute for Life Sciences, University of Ulsan College of Medicine, and Yeungnam University animal facility. The animal procedures were approved by the Institutional Ethics Committee and Institutional Animal Care Committee of the University of Ulsan College of Medicine (2019-12-218, 2019-12-316) and Yeungnam University (2020-030). All animal studies were conducted in a double-blind manner by separating animal breeding and tissue analysis. Mouse experiments were repeated twice, and the numbers of animals in the experimental groups are described in detail in the Figures.
2.2. Human Blood Samples
This study was conducted according to the principles of the Declaration of Helsinki. Thirty cc of whole peripheral blood was obtained from patients with systemic lupus erythematosus (SLE) and healthy controls at Seoul St. Mary’s Hospital. The enrollment criteria for patients with lupus included those age 20–60 years who had been followed in the rheumatology clinic for their diagnosis of SLE per the American College of Rheumatology criteria [
15]. The enrollment criteria for healthy controls included males or females aged between 20 to 60 years without autoimmune or inflammatory diseases. We excluded patients with any serious systemic illness, including infection and cancer. Written informed consent was obtained from all patients, and the Institutional Review Board of Seoul St. Mary’s Hospital, in Seoul, Korea, approved the study (IRB number: KC20TISI0491).
2.3. Antibodies and Reagents
Functional grade anti-CD3 (145-2C11), anti-CD28 (37.51), anti-IL-4 (11B11), and anti-IFN-γ (H22) were purchased from BioXCell (West Lebanon, NH, USA). Fluorescently labeled anti-B220 (RA3-6B2), anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD25 (PC61), anti-NK1.1 (PK136), and anti-TCR-β (H57-597) were purchased from BioLegend (San Diego, CA, USA). Fluorescently labeled anti-phospho-c-Jun (Ser63) (KM-1) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-ATF4 (D4B8), anti-GRP78, anti-PERK (C33E10), anti-phospho-PERK (16F8), anti-eIF2a (D7D3), anti-phospho-eIF2a (Ser51) (D9G8), anti-phospho-p65 (Ser536) (93H1), anti-IκBα, anti-phospho-IκBα (Ser32), and anti-phospho-Erk1/2 (Thr202/Tyr204) (D13.14.4E) were provided by Cell Signaling Technologies (Danvers, MA, USA). FAT10, UBA6, and USE1 antibody were described previously [
9,
10,
13]. The recombinant murine IL-1β (rmIL-1β), IL4, IL-6, IL-12p70, IL-23, and TGF-β1 were purchased from BioLegend (San Diego, CA, USA).
2.4. Flow Cytometry Analysis
Single-cell suspensions from the thymus, lymph node, spleen, lung, or liver were prepared and resuspended in staining buffer (PBS containing 1% FBS and 2 mM EDTA) and stained with the indicated antibodies (Abs). Cells were incubated with PMA plus ionomycin for 4 h, and GolgiStop (BD Bioscience, San Jose, CA, USA) was added 2 h before harvesting the cells to detect cytokines. To detect transcription factors (phospho-p65 and phospho-c-Jun), we followed the manufacturer’s protocol from eBioscience (San Diego, CA, USA). Data were acquired by NovoCyte (ACEA Biosciences Inc., San Diego, CA, USA) and analyzed with NovoExpress (ACEA Biosciences Inc., San Diego, CA, USA).
2.5. T Cell Isolation and Activation
Naïve-enriched CD4 or CD8 T cells were purified by negative selection using CD4+ or CD8+ T cells isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany). For in vitro studies, isolated CD4 or CD8 T cells (1 × 106 per well in a 24-well plate) were stimulated with a plate coated in 5 μg/mL of anti-CD3 mAb plus anti-CD28 mAb for 3 days. Splenocytes were stimulated with soluble anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) for 3 days to differentiate T cells. Cytokines or cytokine-blockade agents were added in culture as indicated. IL-1β, IL-4 IL-6, IL-12, IL-23, and TFG-β were used at concentrations of 10 ng/mL, 10 ng/mL, 50 ng/mL, 50 ng/mL, 20 ng/mL, and 20 ng/mL, respectively. Anti-IL-4 and anti-IFN-γ, as well as their respective control IgGs, were all used at 10 μg/mL. All cell cultures were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol.
2.6. In Vivo T Cell Stimulation by Anti-CD3 Antibody
C57BL/6 mice were injected with 20 μg of anti-CD3 Abs. Twenty-four h after injection, mice were euthanized and splenocytes were isolated and restimulated in vitro for 4 h with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (1 μM; both from Calbiochem, La Jolla, CA), with addition of monensin (eBioscience) during the final 2 h. Cells were then stained for intracellular cytokines and surface markers using the intracellular cytokine staining kit (BioLegend).
2.7. Induction of Multi-Organ Inflammation in RAG1-Knock out Mice
The CD3+CD25−CD122− T cells from C57B/6 or UBA6D mice splenocytes were isolated using a mouse pan T cell isolation kit (Miltenyi Biotec) with biotin-conjugated anti-CD25 and anti-CD122 Abs. The CD3+CD25−CD122− cells (1 × 106/100 μL) were transferred into 6–8-week-old RAG1-KO mice via intravenous injection. After 2 weeks, the mice received 20 μg of polyinosinic:polycytidylic acid (poly I:C) three times per week for an additional 2 weeks. Mice were then euthanized, and organs were collected for analysis.
2.8. Preparation of Lung and Liver Cell Suspension
Mice lungs and liver were dissected into small fragments, placed in a grinder, and processed in a tissue homogenizer. Tissue homogenates were filtered through a 100 μm nylon mesh, washed twice in PBS, and resuspended in culture medium.
2.9. Histology and Immunofluorescence Staining
Lung, liver, and colon samples were fixed with 4% paraformaldehyde and embedded in paraffin. The tissues were then sectioned into 5 μm slices and stained with hematoxylin and eosin (H&E) after deparaffination and rehydration.
2.10. Human T Cell Analysis
Peripheral blood mononuclear cells (PBMCs) were prepared from the whole blood of healthy or SLE donors using a density gradient centrifugation method with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Cells were then stained with anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 (SK1). Some methods required that CD4 or CD8 T cells be isolated from PBMCs using a human CD4 or CD8 T cell isolation kit (Miltenyi Biotec). The cells were then stimulated with pre-coated anti-CD3/28 mAb for 3 days. Intracellular production levels of IFN-γ were measured as described above.
2.11. Immunoblotting
Whole-cell lysates were prepared in ice-cold RIPA lysis buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and a phosphatase inhibitor cocktail (Pierce). Protein concentration was measured using the BCA method (Pierce). After boiling in loading buffer, lysates were separated on SDS-PAGE and analyzed by western blotting using the following antibodies: (phospho-IκBα, IκBα), ATF4, (phosphor-PERK, PERK), (phosphor-eIF2, eIF2), GRP78, UBA6, USE1, and Fat10. The membrane was stripped and reprobed with α-tubulin Ab (Abcam, Cambridge, UK) or UBA6-specific polyclonal Ab, generated as described previously [
7]. Horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary Abs (GE Health, Chicago, Illinois, USA) and ECL (GE Health) were used to detect bound Abs.
2.12. Quantitative PCR
RNA was isolated by Trizol or easy-BLUE extraction according to the manufacturer’s instructions (Invitrogen and iNtRON Biotechnology, Seongnam, Korea). CDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen and iNtRON Biotechnology) and subjected to quantitative real-time PCR using SYBR Green I Master Mix on a CFX Connect Real-Time PCR system (Bio-Rad, CA, USA). Each value was normalized to human β-actin or GAPDH. The specific primers used for PCR amplification were previously described [
15].
2.13. Nuclear NF-κB p65 Activation Assay
According to the manufacturer’s protocol, the nuclear NF-κB p65 activity was quantified using an ELISA-based TransAM NF-κB p65 kit (Active Motif, Carlsbad, CA, USA). In brief, whole cell protein extracts (10 µg per well) were added to a 96-well plate containing an immobilized oligonucleotide with an NF-κB element for 1 h incubation at room temperature, during which activated NF-κB p65 could bind specifically to the oligonucleotide. NF-κB p65 Ab (100 µL, at 1:1000 dilution) was then added to each well for 1 h followed by 100 µL of anti-rabbit HRP-conjugated Ab (1:1000 dilution) for 1 h. After washing three times in wash buffer, developing solution (100 µL) was added for up to 15 min, and the colorimetric reaction was stopped after 5 min. The NF-κB p65 activity was determined by reading absorbance on a spectrophotometer (Molecular Devices, CA, USA) at 450 nm with a reference wavelength of 655 nm.
2.14. Statistical Analysis
All statistical analyses were performed using Prism software, version 5 (GraphPad, San Diego, CA, USA). Results are presented as mean ± SEM and a one- or two-way ANOVA (Tukey multiple comparison test) were used for analysis of the data sets. Asterisks denote the level of significance (* p < 0.05, ** p < 0.01), and a p-value of < 0.05 was considered statistically significant.
4. Discussion
This study demonstrates the important role of UBA6 in controlling IFN-γ production in CD4 and CD8 T cells. UBA6 deficiency in T cells resulted in the hyperactivity of IFN-γ production in both CD4 and CD8 T cells, contributing to exacerbation of multi-organ inflammation in RAG1-KO mice. Moreover, UBA6 levels were considerably lower in T cells from patients with SLE compared with those of healthy donors, which negatively correlated with IFN-γ production in T cells in response to stimulation. In the mechanism study of UBA6 in T cell activation, deficiency of UBA6 in T cells led to increased phosphorylation and degradation of IκBα, which led to increased activation of NF-κB p65. Our results provide insight into the role of UBA6 in T cell biology. Our work has identified the important role of UBA6 in T cells and demonstrated UBA6-mediated protein degradation as a mechanism for the regulation of IFN-γ production and exacerbation of autoimmune diseases.
Although some E3 ligases are reported to be critical to maintain peripheral T cell tolerance and their deficiency results in severe autoimmunity, we did not observe any sign of spontaneous autoimmune disease development in UBA6 T cell deficient mice. This may be attributed to the maintenance of functionally active mice (
Supplementary Figure S2). Therefore, naïve T cells transferred to RAG1-KO mice without regulatory T cells developed multi-organ inflammation, likely reflecting that these E3s use different E2s under the control of charging by E1 enzyme. However, like Cbl-b, Itch, and Peli1-deficient T cells, UBA6 deficiency in T cells leads to their hyperactivation, suggesting that both the canonical UBA1 pathway and the non-canonical UBA6 pathway function within the immune system.
SLE is associated with aberrant T cell function, dysfunction of T cell subsets, and imbalance of helper and regulatory T cells. As the systemic autoimmune process of mice lacking UBA6 is similar to that of patients with SLE, we suspected that dysfunction in the ubiquitin pathway is likely related to the pathogenesis of SLE. Elevated expression of IFN is present in SLE, and the IFN signature is the most prevalent molecular pathway activated in SLE. Additionally, the transcription factor NF-κB, which is activated by IFN and regulates IFN genes [
19], is reportedly involved in SLE [
20]. Our current study found that UBA6 expression markedly decreased in peripheral blood samples of patients with SLE compared with that of healthy controls. Patients with lupus also had increased expression of IFN-γ positive cells, and UBA6 expression was negatively correlated with IFN-γ positive cells. Patients with lupus express higher levels of NF-κB than healthy controls. Our result suggests that the known aberrant T cell subsets in SLE are likely to be partially related to dysfunction or deficiency of UBA6 due to its decreased T cell regulatory function.
TCR stimulation by antigen recognition induces transcription factors on T cells, including NF-κB, nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1). Activation of these transcription factors involves proliferation and activation of T cells and their differentiation to Th1, Th2, and Th17 cells. UBA6-deficiency promoted over activation of NF-κB, the primary transcription factor for Th1 and Th17 differentiation. However, UBA6 is only upregulated during differentiation of Th1, but not Th2 and Th17 cells, indicating that UBA6 may control the tolerance of Th1 immune responses by suppression of NF-κB activation. In addition, previous work has shown that deletion of UBA6 in other cell types such as fibroblasts inhibits MAPK (Erk1/2 and JNK1/2) activation [
10]. In contrast, our data showed that deletion of UBA6 in T cells did not affect MAPK activation. Thus, the requirement for UBA6 in the MAPK and NF-κB pathways may depend on the cell type and stimuli.
NF-κB signaling is well established as a critical step in many biological processes in the immune system, including inflammation, immunity, cell survival [
14,
21], and immune cell development [
22]. Moreover, NF-κB is a central regulator of T cell survival, proliferation, and effector function during T cell activation. Although NF-κB activation in CD4
+ T cells has been studied extensively, little is understood about its signaling in CD8
+ T cells. It has been reported that certain modifications (phosphorylation of Ser311 and acetylation of Lys310) of NF-κB p65 are defective in anergic CD8 T cells, whereas early NF-κB activation events including IκB degradation and NF-κB nuclear translocation occur normally [
23]. In addition, unlike CD4
+ T cells, in which TCR downstream molecules (PKCθ, Bcl10, and Malt1) are essential for TCR-induced NF-κB activation, CD8
+ T cells possess an alternative NF-κB activation that is independent of PKCθ, Bcl10, and Malt1 [
24]. This study found that UBA6 is a novel negative regulator of NF-κB activation in CD3 T cells. Further work is required to determine the precise molecular mechanisms underlying NF-κB activation by UBA6, and to examine whether USE1 functions together with UBA6 in this context. Nevertheless, our study indicates that both the canonical and noncanonical arms of the UPS system function separately, and likely cooperatively, to modulate the immune system.